Journal: ACS Bio & Med Chem Au

Article Title: Discovery of Small-Molecule Antagonists of PHF1 and 19 Demonstrates the Ligandability of PRC2 Accessory Proteins

doi: 10.1021/acsbiomedchemau.5c00150

Figure Lengend Snippet: UNC7242 ( 20 ) is unable to displace recombinant or endogenous PHF1 or 19 from nucleosomes. (a) SDS-PAGE gel demonstrating pull-down of recombinant PHF1 (Tudor + PHD1 + PHD2) (left) and PHF19 (Tudor + PHD1) (right) by biotinylated H3K36me3 nucleosomes in the presence and absence of UNC7242 ( 20 ). (b) Western blot analysis of Jurkat N6 cell lysates biochemically separated into chromatin bound and unbound fractions after treatment with 100 μM UNC7242 ( 20 ), compound 22 , and nonbinding negative control, compound 7 .

Article Snippet: Twenty μg biotinylated H3K36me3 nucleosomes (EpiCypher, 16–0320) were immobilized on 30 μL Dynabeads MyOne streptavidin T1 magnetic beads (Invitrogen) precleared with binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 10% glycerol, 0.1% NP-40 (IGEPAL), 1 mM DTT).

Techniques: Recombinant, SDS Page, Western Blot, Negative Control

Journal: Molecular cell

Article Title: Chromatin-bound U2AF2 splicing factor ensures exon inclusion

doi: 10.1016/j.molcel.2025.04.013

Figure Lengend Snippet: (A) Browser track showing raw counts for RNAPIIS5P and RNAPIIS2P and spike-in calibrated counts for U2AF2. (B–D) Heatmaps (bottom) and average plots (top) showing raw counts for RNAPIIS5P (B), RNAPIIS2P (C), and spike-in calibrated counts for U2AF2 (D) on U2AF2 chromatin-bound genes under the indicated treatments. See also . (E) RNAPIIS2P density on chromatin-bound or RNase A-sensitive genes. RNAPIIS2P RPK = total RNAPIIS2P mapped fragment number on regions including 1 kb upstream and downstream of the gene (gene length in kilobases). RNAPIIS2P density is log 2 (1 + RNAPIIS2P RPK). (F) Nascent transcript levels from chromatin-bound and RNase A-sensitive genes were quantified using published transcripts per million (TPM) values from chromatin fraction total RNA-seq data. (G) Scatterplots showing positive correlations between the log 2 -transformed total mapped fragment numbers of H3K36me3 and U2AF2 on gene bodies. Each dot represents a single gene. (H) Browser track showing raw counts for H3K36me3 and spike-in calibrated counts for U2AF2. (I and J) Heatmaps (bottom) and average plots (top) showing raw counts for H3K36me3 (I) and spike-in calibrated counts for U2AF2 (J) on U2AF2 chromatin-bound genes under the indicated treatments.

Article Snippet: We used the following antibodies: Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) (Antibodies Online, Cat# ABIN101961), H3K36me3 (Rabbit monoclonal anti-H3K36me3, Epicypher, Cat# 13-0058), RNAPIIS2P (Rabbit monoclonal anti-RNAPIIS2P, Cell Signaling Technology, Cat# 13499S), RNAPIIS5P (Rabbit monoclonal anti-RNAPIIS5P, Cell Signaling Technology, Cat# 13523S), SF3B1 (Rabbit monoclonal anti-SF3B1, Cell Signaling Technology, Cat# 14434S), U2AF1 (Rabbit polyclonal anti-U2AF35, Bethyl Laboratories, Cat# A302-079A) and U2AF2 (Rabbit polyclonal anti-U2AF65, Abcam, Cat# ab37530), SRSF1 (Rabbit polyclonal anti-SRSF1, Thermo Fisher Scientific, Cat# A302-052A) and SRSF3 (Rabbit polyclonal anti-SRSF3, MBL International, Cat#RN080PW).

Techniques: RNA Sequencing, Transformation Assay

Journal: Molecular cell

Article Title: Chromatin-bound U2AF2 splicing factor ensures exon inclusion

doi: 10.1016/j.molcel.2025.04.013

Figure Lengend Snippet: (Top) RNAPII interacts directly with U1 snRNP, enabling the rapid recognition of the 5′ splice site in nascent pre-mRNA by U1 snRNP. U2AF2 initially binds to the RNAPII CTD , and transfers from elongating RNAPII to chromatin through the H3K36me3 mark deposited by SETD2 during transcription elongation. (Middle panel) U2AF2 detaches from chromatin and transfers to the polypyrimidine tract on nascent pre-mRNA, stabilizing U2AF1 at the 3′ splice site and SF3B1 at the branchpoint to form the spliceosomal A complex. (Bottom) Recruitment of the preassembled U4/U6.U5 tri-snRNP complex forms the pre-catalytic B complex, the first fully assembled spliceosome with all five snRNPs. , The B complex then undergoes extensive rearrangements to activate the spliceosome for splicing. , Overall, U2AF2 binding to chromatin guides the spliceosome in recognizing splice junctions, ensuring efficient exon inclusion.

Article Snippet: We used the following antibodies: Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) (Antibodies Online, Cat# ABIN101961), H3K36me3 (Rabbit monoclonal anti-H3K36me3, Epicypher, Cat# 13-0058), RNAPIIS2P (Rabbit monoclonal anti-RNAPIIS2P, Cell Signaling Technology, Cat# 13499S), RNAPIIS5P (Rabbit monoclonal anti-RNAPIIS5P, Cell Signaling Technology, Cat# 13523S), SF3B1 (Rabbit monoclonal anti-SF3B1, Cell Signaling Technology, Cat# 14434S), U2AF1 (Rabbit polyclonal anti-U2AF35, Bethyl Laboratories, Cat# A302-079A) and U2AF2 (Rabbit polyclonal anti-U2AF65, Abcam, Cat# ab37530), SRSF1 (Rabbit polyclonal anti-SRSF1, Thermo Fisher Scientific, Cat# A302-052A) and SRSF3 (Rabbit polyclonal anti-SRSF3, MBL International, Cat#RN080PW).

Techniques: Binding Assay

Journal: Cell Reports Medicine

Article Title: Histone lysine demethylase 4 family proteins maintain the transcriptional program and adrenergic cellular state of MYCN-amplified neuroblastoma

doi: 10.1016/j.xcrm.2024.101468

Figure Lengend Snippet: QC6352 impacts the chromatin accessibility of ADRN CRC TFs in MYCN -amplified cells (A and B) Adrenergic or mesenchymal median score in response to DMSO or 100 nM of QC6352 treatment for 48 h in adrenergic dominant cell lines (BE2C, SIMA, and KELLY) (A) and mesenchymal dominant cell lines (SK-N-AS and SK-N-SH) (B). KDM4 inhibition results in enhanced MES scoring with reduced ADRN signatures in all tested cell lines (red arrow indicates direction of signature change). (C) HOMER Motif analysis showing the predicted adrenergic CRC TF binding motifs in which genome-wide chromatin accessibility is impacted by QC6352 treatment of SIMA cells. (D) PHOX2A, ASCL1, and MYCN motif densities around ATAC-seq open chromatin regions (±1,000 bp) by categories in SIMA cells. Motif density was determined by the HOMER program and normalized to that in a background sequence of equal length. (E) STRING network analysis of transcriptional factors predicted to bind the regions with reduced chromatin accessibility by QC6352 treatment of SIMA cells. The expression threshold for genes encoding TFs is set as log2CPM > 1. CPM, counts per million mapped reads. Red color indicates ADRN CRC TFs. Blue color indicates MES CRC TFs. (F) IGV snapshots show chromatin accessibility at MYCN , HAND2 , PHOX2B , and ALK loci is reduced by QC6352 in SIMA and BE2C cells. (G) Density heatmaps for CUT&TAG of KDM4A-C genomic binding in BE2C cells, and ChIP-seq density heatmaps of the indicated histone marks in BE2C cells after 48 h treatment with control or 200 nM QC6352, ranked by MYCN read intensity within ± 5 kb of peak summits. MYCN ChIP-seq data were retrieved from GEO SuperSeries GSE94824. (H) The indicated epigenetic marks upregulated and downregulated by QC6352 treatment of BE2C cells for 48 h followed by ChIP-seq or CUT&TAG analyses. (I) The global distribution of H3K9me3, H3K36me3, H3K27Ac, H3K4me1, and H3K27me3 peaks at different regions of annotated genes. 3′ UTR, 3′ untranslated region; 5′ UTR, 5′ untranslated region; TSS, transcription start site. (J) Snapshot of using the IGV program displaying the KDM4s, H3K9me3, H3K36me3, H3K27me3, H3K27Ac, and H3K4me1 peaks at the PHOX2B and ASCL1 genomic loci.

Article Snippet: The primary antibodies H3K36me3 (RevMab, 31-1051-00), H3K4me1 (Abcam, ab8895) and H3K27ac (Abcam, ab4729) were coupled with 25 μL magnetic beads (Dynabeads M-280 Sheep Anti-Rabbit IgG, Invitrogen 11203D) while H3K9me3 (Absolute, 309M3-B) antibodies were coupled with 100μL of Dynabeads 280 streptavidin overnight in the cold room at 4°C, washed three times with PBS/BSA (1X PBS/5 mg/mL BSA (fraction V)) at 4°C, then incubated with sonicated DNA chromatin samples overnight in the cold room at 4°C.

Techniques: Amplification, Inhibition, Binding Assay, Genome Wide, Sequencing, Expressing, ChIP-sequencing, Control

Journal: Cell Reports Medicine

Article Title: Histone lysine demethylase 4 family proteins maintain the transcriptional program and adrenergic cellular state of MYCN-amplified neuroblastoma

doi: 10.1016/j.xcrm.2024.101468

Figure Lengend Snippet:

Article Snippet: The primary antibodies H3K36me3 (RevMab, 31-1051-00), H3K4me1 (Abcam, ab8895) and H3K27ac (Abcam, ab4729) were coupled with 25 μL magnetic beads (Dynabeads M-280 Sheep Anti-Rabbit IgG, Invitrogen 11203D) while H3K9me3 (Absolute, 309M3-B) antibodies were coupled with 100μL of Dynabeads 280 streptavidin overnight in the cold room at 4°C, washed three times with PBS/BSA (1X PBS/5 mg/mL BSA (fraction V)) at 4°C, then incubated with sonicated DNA chromatin samples overnight in the cold room at 4°C.

Techniques: Recombinant, Modification, Synthesized, Saline, Transfection, Apoptosis Assay, Purification, Transgenic Assay, Control, Expressing, Plasmid Preparation, Software